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US20030092692A1 - Cytoprotective steroids (II) - Google Patents

Cytoprotective steroids (II) Download PDF

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US20030092692A1
US20030092692A1 US10/203,880 US20388002A US2003092692A1 US 20030092692 A1 US20030092692 A1 US 20030092692A1 US 20388002 A US20388002 A US 20388002A US 2003092692 A1 US2003092692 A1 US 2003092692A1
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hydroxy
composition
substituted
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Richard Lathe
Jonathan Seckl
Keith Martin
Ernst Wulfert
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/567—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in position 17 alpha, e.g. mestranol, norethandrolone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the use of known and novel steroids in the treatment of conditions that lead to damage and death of cells, particularly of nerve cells and more particularly of CNS nerve cells.
  • the use particularly relates to treatment of acute conditions affecting the CNS, such as stroke and head or spinal trauma, wherein treatment within days, or even hours, of onset of the condition is required to prevent irreversible damage and/or death.
  • estradiol ie. 3 ⁇ -, 7 ⁇ -, 17 ⁇ -estradiol, and their 7-keto precursors have been known for many years.
  • U.S. Pat. No. 2,418,603 describes the use of 7 ⁇ -hydroxy and/or 7-keto-estradiol in the synthesis of estradiol and ascribes to them at most ⁇ fraction (1/300) ⁇ of the estrogenic activity of estrone.
  • the 7 ⁇ -hydroxy compounds at least are known to be a naturally occurring metabolites of estrogens and related compounds (see Holler et al (1984) J. Steroid Biochem Vol 20, No 3 pp785-787).
  • transcripts of the enzyme CYP7B are reduced in Alzheimer's disease and that this enzyme has activity in converting DHEA, pregnenolone and estradiol to their corresponding 7 ⁇ -hydroxy derivatives.
  • Relative activity of the enzyme against substrates is in the order DHEA>pregnenolone>estradiol, with the implication being that greater effect of enzyme depletion would be seen on the antiglucocorticoids 7 ⁇ -hydroxy DHEA and 7 ⁇ -hydroxy-pregnenolone, with resultant reduction in protection against deleterious effects of cortisol (see U.S. Ser. No. 09/168,218 from WO97/37664).
  • 7 ⁇ -hydroxy estradiol and precursors thereof whose synthesis would also be impaired by such enzyme loss, should also be used in treating Alzheimer's disease, although no particular mechanism is implicated for its actions.
  • WO 99/52532 claims use of DHEA and its congeners in general in treating stroke and CNS trauma, but of 54 congeners described as being useful, none is a 7 ⁇ -hydroxy or 7-keto steroid.
  • WO 98/22113 claims use of estrogens in treatment of ischemia, whether CNS or peripheral, but only exemplifies use of 17 ⁇ - and 17 ⁇ -estrogens that are unsubstituted at the 7-position.
  • the present inventors have now surprisingly determined that 7 ⁇ -hydroxy DHEAs, 7 ⁇ -hydroxypregnenolones, 7 ⁇ -hydroxyestradiols and their metabolic precursors may be used to treat acute neurodegenerative disease states, such as stroke and head trauma, as well as in the known chronic and anti-glucacorticoid applications already described. Furthermore, far from being a relatively unimportant steroid metabolite physiologically, not only does 7 ⁇ -hydroxy estradiol have a significant therapeutic role in chronic diseases such as Alzheimer's, it also has a surprising neuroprotective efficacy applicable to treating these acute disorders and disease states. This is particularly surprising, as the inventors do not find this efficacy to be shared by the 7-unsubstituted forms DHEA, pregnenolone or estradiol themselves, which are otherwise thought to be protective in chronic disease.
  • a first aspect of the present invention provides a method of treating a patient in need of therapy for acute cellular degeneration due to metabolic compromise of cells comprising administering to that patient a therapeutically effective amount of a 7 ⁇ -hydroxy substituted steroid selected from 7 ⁇ -hydroxy-estradiols, 7 ⁇ -hydroxy-dehydroepiandrosterones, 7 ⁇ -hydroxy-pregnenolones and metabolic precursors of any of these.
  • a second aspect of the invention comprises the use of a 7 ⁇ -hydroxy substituted steroid selected from 7 ⁇ -hydroxy-estradiols, 7 ⁇ -hydroxy-dehydroepiandrosterones, 7 ⁇ -hydroxy-pregnenolones and metabolic precursors of any of these for the manufacture of a medicament for the treatment of acute cellular degeneration due to metabolic compromise of cells.
  • a third aspect of the present invention provides a cell function protectant composition characterised in that it comprises a 7 ⁇ -hydroxy substituted steroid selected from 7 ⁇ -hydroxy-estradiols, 7 ⁇ -hydroxy-dehydroepiandrosterones, 7 ⁇ -hydroxypregnenolones and metabolic precursors of any of these together with a pharmaceutically acceptable carrier in a form suitable for parenteral administration, particularly in an injectable form, eg suitable for intravenous infusion.
  • a pharmaceutically acceptable carrier in a form suitable for parenteral administration, particularly in an injectable form, eg suitable for intravenous infusion.
  • Such composition may for example be administered as an aqueous solution through a catheter, with possibility of providing it with other fluids to a patient eg. suffering from stroke or head or spinal trauma.
  • Preferred methods and compositions of the invention are directed at treating acute neuronal degeneration, such as occurs after head trauma or an ischemic focus within the CNS.
  • Preferred metabolic precursors are those which are converted directly to 7 ⁇ -steroid by removal of one or more alkyl, acyl, phosphonyl or sulphoxyl groups from one or more of the three —OH groups present on such steroids or by conversion of a 7-keto form.
  • the steroid in each case is 7 ⁇ -hydroxy-estradiol (ie. 3 ⁇ -, 7 ⁇ -, 17 ⁇ -estriol, also estra-1,3,5(10)-triene-3 ⁇ , 7 ⁇ , 17 ⁇ -triol) or its 7-keto analogue, or is 7 ⁇ -hydroxy-DHEA or 7-keto-DHEA or carboxylic acid esters of either, eg. 3-acetyloxy. 7 ⁇ -hydroxy-pregnenolone is found to be less active than these in the present method and use
  • alkyl ethers or carboxyl, phosphonyl or sulphonyl esters are preferred metabolic precursors for the method, use or composition of the invention.
  • Alkyl is preferably C 1-6 alkyl.
  • Preferred 7 ⁇ -hydroxy-estradiol type compounds for the method, use and composition of the invention are of general formula Ia or Ib set out below
  • OR 1 , OR 2 and OR 3 each independently represents a free hydroxy group, an ether group or an esterified hydroxy group and R 4 is hydrogen, substituted or unsubstituted C 1-6 alkyl, C 2-6 alkenyl or C 2-6 alkynyl, more preferably hydrogen or ethynyl.
  • R 1 , R 2 and R 3 are independently selected from substituted or unsubstituted C 1-6 alkyl groups, any such substituents being selected from OH, halogen (F, Cl, Br, I), amino, C 1-6 alkylamino, C 1-6 dialkylamino, —COOH or —COOR 5 wherein R 5 represents a C 1-6 alkyl group which may be unsubstituted or substituted by one of the substituents referred to above; or —OR 1 , —OR 2 and —OR 3 each independently represents an esterified hydroxy group, of the formula R 6 COO—, wherein R 6 may be selected from substituted or unsubstituted C 1-6 alkyl groups, any such substituents being selected from —OH, halogen (F, Cl, Br, I), amino, C 1-6 alkylamino, C 1-6 dialkylamino, —COOH or —COOR 5 wherein R 5 represents a C 1-6 alkyl groups, any such substitu
  • —OR 1 , —OR 2 and —OR 3 each independently represents an esterified hydroxy group of formula —OP(OH) 3 , or a sulphate group.
  • acute cellular degeneration is meant a condition in which cells are losing functionality, temporarily or irreversibly and/or in a process of dying whether through apoptosis or other mechanism.
  • metabolic compromise an acute state where a cell is either cut off from sufficient oxygen and/or energy source, eg. a metabolisable molecule that may be used by the cell to produce energy in the form of ATP, NADH and NADPH, most particularly being glucose, or is rendered incapable of using such oxygen or molecule by mechanical injury, eg. resulting in inflammation, increase of intracranial pressure and neuron degeneration eg. apoptosis.
  • energy source eg. a metabolisable molecule that may be used by the cell to produce energy in the form of ATP, NADH and NADPH, most particularly being glucose, or is rendered incapable of using such oxygen or molecule by mechanical injury, eg. resulting in inflammation, increase of intracranial pressure and neuron degeneration eg. apoptosis.
  • this compromise is that which is acutely life threatening, with treatment required within hours, or at most a few days, to prevent nerve cell death, whether by apoptosis or other mechanism.
  • Coma may for example be that brought on by an anoxic episode, such as due to drowning, asphyxiation or cessation of breathing or other condition resulting in failure of the cerebral circulation.
  • the treatment may be provided prophylactically, such as where a patient is at risk of having an ischemic event, eg. stroke, or may be provided immediately or as soon after the onset of the condition as possible.
  • ischemic event eg. stroke
  • Other examples of risk states include diagnosis of diseased blood vessels, eg. atherosclerosis or where it is necessary to perform coronary surgery, both benefiting from treatment for sequelae of ischemic nature as opposed to just anticoagulant therapy.
  • compositions for use in the may be administered by any conventional method including enteral (for example oral and rectal) or parenteral (for example delivery into the nose or lung or injection into the veins, arteries, brain, spine, peritoneum, muscles or sub-cutaneous region. Most preferably the compounds are administered by enteral (ie. oral), intravenous or transdermal administration
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • the dosage will be determined by the physician but may be between 0.01 mg and 1.0 g/kg/day, for example between 0.1 and 500 mg/kg/day.
  • the compound can be administered at 1.0 mg to 1.5 g per m 2 per day, for example 3.0-200.0 mg/m 2 /day.
  • a compound of the invention Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the formulations may conveniently be presented in unit dosage form.
  • a unit dosage form may comprise for example 2.0 mg to 2.0 g, for example 5.0 mg to 300.0 mg of active ingredient.
  • Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • FIG. 1 Shows a bar chart indicating the % cells damaged in CA1 after 180 minutes oxygen withdrawal followed by 24 hours of reperfusion, ie. hypoxic challenge, in presence of stated doses of 7 ⁇ -OHDHEA and 7 ⁇ -OHE2.
  • FIG. 2 Shows a bar chart illustrating the dose dependent effect of 7 ⁇ -OHDHEA and 7 ⁇ -OHE2.
  • FIG. 3 Shows a bar chart illustrating the effect of 7 ⁇ -OHE2 on cell death in glutamate toxicity assay of primary cortical neurones. Estradiol was found to have no effect.
  • FIG. 4 Shows a bar chart illustrating the effect of 7 ⁇ -OHDHEA 7 ⁇ -OHE2 on cell death in glutamate toxicity assay of primary cortical neurones.
  • the title compound can be prepared by a two step process involving epoxidation of estra-1,3,5(10),6-tetraene-3,17 ⁇ -diol diacetate (7), followed by opening of the epoxide ring, with removal of the acetate groups, by reaction with lithium aluminium hydride.
  • the following protocol was used due to temporary problems in sourcing estratetraene (7), making it necessary to undertake 6 extra steps to prepare (7) from ⁇ -estradiol (1).
  • ⁇ -Estradiol (1) was converted to the diacetate (2) in 94% yield by reaction with acetic anhydride heated under reflux for 1 hour in pyridine.
  • the diacetate (2) was oxidised with chromium (VI) oxide in acetic acid-water, followed by purification by chromatography and recrystallisation from ethanol-water to afford 6-keto-estradiol diacetate (3) in 18% yield (23% yield for small scale reaction) 2 .
  • the ketone (3) was reacted with tosylhydrazide heated under reflux for 6 h in ethanol to produce the tosylhydrazone (4) in 83% yield.
  • the acetate groups were removed in quantitative yield by reaction at room temperature with methanolic potassium hydroxide.
  • the tosylhydrazone diol (5) was then converted into the estratetraene (6) in 36% yield after purification by flash chromatography (54% yield for small scale reaction), by a Shapiro reaction in which a solution of (5) in dry tetrahydrofuran was treated with methyllithium at 0° C. and then allowed to warm to room temperature. Reaction of the diol (6) with acetic anhydride heated under reflux for 1 h in pyridine gave the diacetate (7) in 91% yield 3 .
  • estratetraene (7) was epoxidised by treatment in ether-tetrahydrofuran with an excess of monoperphthalic acid 4 at 5° C. for 1 day.
  • An 85% yield of the 6 ⁇ ,7 ⁇ -epoxide (8) was obtained following purification by flash chromatography.
  • the synthesis was completed by reaction of the epoxide diacetate (8) with lithium aluminium hydride heated under reflux in tetrahydrofuran. After purification by flash chromatography, an 85% yield of 7 ⁇ -hydroxyestradiol (9) was obtained 5 .
  • the 1 H NMR, IR and mass spectra of (9) were consistent with its structure
  • step 1 33% EtOAc: (1) R f 0.3, (2) R f 0.9; step 2, 20% EtOAc: (2) R f 0.8, (3) R f 0.6; step 3, 20% EtOAc: (3) R f 0.6, (4) R f 0.4; step 4, 33% EtOAc: (4) R f 0.6, (5) R f 0.4; step 5, 40% EtOAc: (5) R f 0.5, (6) R f 0.2; step 6, 40% EtOAc: (6) R f 0.2, (7) R f 0.6; step 7, 25% EtOAc: (7) R f 0.5, (8) R f 0.3; step 8, 8% MeOH: (8) R f 0.85, (9) R f 0.15
  • Acetic anhydride (2.8 ml) was added to a stirred solution of estra-1,3,5(10),6-tetraene-3,17 ⁇ -diol (6) (0.80 g, 2.96 mmol) in dry pyridine (12 ml) and the mixture was heated under reflux for 1 hour. When cool, the mixture was poured into ice-water (40 ml) with stirring. The solid was collected by filtration, washed with cold water and dried in vacuo (ca. 60° C.) for 1 hour to afford the product as a beige solid (0.95, 91%).
  • a suspension of phthalic anhydride (10.0 g, 67.5 mmol) and sodium perborate (14.9 g, 96.8 mmol) in water (45 ml) was stirred at 0° C. for 2 hours.
  • the mixture was filtered, the filtrate was acidified with ice-cold 30% sulphuric acid and extracted with ether (3 ⁇ 25 ml). Emulsion formation was a problem, so some brine was added.
  • the combined ethereal solution was washed with brine and dried (MgSO 4 ).
  • the ethereal solution of monoperphthalic acid was filtered and stored at 5° C. It should be ca. 0.7M monoperphthalic acid.
  • Ethereal monoperphthalic acid solution 14 ml was added to a stirred solution of estra-1,3,5(10),6-tetraene-3,17 ⁇ -diol diacetate (7) (0.84 g, 2.37 mmol) in 1:1 ether-tetrahydrofuran (24 ml) at 0° C. The solution was transferred to a refrigerator and left to stand at 5° C. for 1 day. Ether (50 ml) was added, the solution was washed with sat. sodium hydrogen carbonate solution (20 ml, 2 ⁇ 10 ml), dried (MgSO 4 ) and evaporated.
  • Wistar rat pups (8-11 days old) were decapitated and the hippocampus rapidly dissected into ice-cold Goy's balanced salt solution supplemented with 4.5 mg/ml glucose. Slices were separated and plated into Millicell CM culture inserts (4 per well) and maintained at 37° C./5% CO 2 for 14 days. Maintenance medium consisted of 25% heat-inactivated horse serum, 25% Hank's balanced salt solution (HBSS) and 50% minimum essential medium with added Earle's salts (MEM) supplemented with 1 mM glutamine and 4.5 mg/ml glucose.
  • HBSS Hank's balanced salt solution
  • MEM minimum essential medium with added Earle's salts
  • hypoxia was induced by transferring cultures to SFM (+PI) which had been saturated with 95% N 2 /5% CO 2 .
  • Culture plates (without lids) were then sealed into an airtight chamber in which the atmosphere was saturated with 95% N 2 /5% CO 2 by continuously blowing through gas at 1000 ml/min for ten minutes before being sealed and placed in the incubator for 170 mins (total time of hypoxia was therefore 180 mins).
  • total time of hypoxia was therefore 180 mins.
  • cultures were returned to normoxic SFM containing PI and placed back in the incubator for 24 hours.
  • Neuronal damage was assessed as described previously (Pringle et al., 1996, 1997) using NIH Image 1.60 running on an Apple IIsi computer. Images were captured using a COHU monochrome camera and saved onto optical disk for offline analysis using NIH image. Light transmission images were captured prior to the addition of drugs, and PI fluorescence images recorded at the end of the 24 hour post-hypoxia recovery period. The areas of the CA1, CA3 and dentate gyrus (DG) cell layers were determined from the transmission image. The area of PI fluorescence in each of the cell layers was measured using the density slice function within NIH Image, and neuronal damage expressed as the percentage of the regional area in which PI fluorescence was detected above background.
  • DG dentate gyrus
  • Steroid compounds were prepared by making an initial 1 mg/ml solution in ethanol and further diluting down in SFM. Compounds were included in the cultures for 45 minutes prior to hypoxia, during the hypoxic episode and during the post-hypoxic recovery period. Control experiments consisted of cultures treated with vehicle alone.
  • L-glutamate is the major excitatory amino acid in the brain of vertebrates and mediates physiological responses such as synaptic plasticity and long term potentiation.
  • overstimulation of glutamate receptors, particularly of the NMDA subtype is believed to initiate cellular processes leading to neurodegenerative conditions.
  • NMDA receptors particularly of the NMDA subtype
  • In vitro a brief exposure to glutamate causes neuronal death mainly by hyperstimulating NMDA receptors present on most neurones causing calcium ion influx.
  • Rat cortical neurons were cultured by modification of the methods of Dichter (1973) Brain Res 149, p279-293 and Durkin et al (1996) J. Neurochem 66, p951-962.
  • a female Wistar rat (Janvier, Le Genest-St-Isle, France: ref RJ WAF151) of 17 days gestation was killed by cervical disclocation, the foetuses removed on embryonoic day 17 and brains removed and placed in ice-cold calcium/magnesium free Hanks balanced salt solution (HBSS: Gibco Life Technologies, Cergy-Pontoise, France: ref 14170-088 batch 302790). Each cortex was dissected and meninges were carefully removed.
  • cortical neurons were dissociated by trypsinization for 5 minutes at 37° C. (trypsin: 0.5 mg/ml: Difco, LPCR, France: ref 0152-14 batch 128856JC) and the reaction stopped by addition of HBSS containing calcium and magnesium (HBSS+ Gibco ref 24020-091; batch 3010598) with 10% of fetal bovine serum FBS; Gibco; ref 1027-098; batch 40Q1681K and 1 mg/ml DNase I (Boehringer Mannheim, Meylan, France; ref 1284938; batch 14880300).
  • the suspension was triturated with a 10 ml pipette (stripette Costar, Dutscher, Brumath, France) and was added in a centrifuge tube (Tube 50 ml Corning, Dutscher) containing 20 ml HBSS ⁇ with 3.5% of BSA (Sigma, L'Isle D'Abeau Chesnes, ref. A6003, batch 29H7606) and centrifuged at low speed (150 ⁇ g) for 10 min (apparatus Sigma 2-15, Bioblock, Illkirch, France).
  • DMEM Dulbecco's Modified Eagle Medium
  • Viable cells were quantified and plated at 35,000/well in 96 well plates (Nunclon, Gibco) previously coated with poly-L-lysine (0.01 mg/ml, France; ref. P5899; batch 28H8504). Cells were allowed to adhere 2 h and maintained in a humidified incubator at 37° C. in 5% CO 2 -95% air atmosphere.
  • Culture medium was then changed to a medium consisting of DMEM, 10% heat-inactivated FBS, 10% heat-inactived horse serum (Gibco, ref. 16505-080; batch 3030347) and 1% L-Glutamine (Gibco, ref. 25030-024; batch 3031499).
  • the cultures were mixed populations containing both neurons and glial cells, to minimize glial growth, the cultures were treated for 2 days with 15 ⁇ g/ml of 5-fluoro-2′-deoxyuridine (Sigma; ref. F8791; batch 97H4048) and 35 ⁇ g/ml of uridine (Sigma, ref. U3003; batch 18H11965) on day 4 of culture.
  • the medium was changed to DMEM/HamF12 (Gibco, ref. 21331-020; batch 23243) supplemented with 12.5 mg/ml glutamine (Gibco ref 15750-032, batch 3009462), 20 ⁇ g/ml transferrin (Sigma, ref. T1428; batch 97H0257), 5 ⁇ g/ml insulin (Sigma, ref. I6634; batch 96H04865), 33 mM glucose (Sigma, ref. G7021; batch 75H07293), 100 ⁇ M putrescin (Sigma, ref. P7505; batch 95H0814), 20 nM progesterone (Sigma, ref.
  • estradiol Sigma, ref. E8875; batch 98H40953
  • DHEA Sigma, ref. D4000; batch 97H0301
  • test compound were added at concentrations of 2, 10, 50 and 100 nM.
  • Human FGF-basic Tebu, Le Perray-en-Yvelines, France; ref. 100-18B; batch 107R0806 E118
  • Glutamate toxicity was evaluated by measuring lactate dehydrogenase (LDH) activity released in the media 24 hours after glutamate exposure, using the CytoTox 96 non-radioactive kit (Promega, Charbonnieres, France, ref. G1780; batches 109578 and 106244L) and quantitated by measuring wavelength absorbance at 450 nm (Labsystems Multiskan Bichromatic, Cergy Pontoise, France).
  • LDH lactate dehydrogenase
  • estradiol and DHEA tested at doses of 0, 50 and 100 nM while failing to protect cortical neurones from glutamate neurotoxicity demonstrated effects in their own right. Both estradiol (10 nM) and DHEA (10 and 50 nM) appeared to protect significantly (p ⁇ 0.05) against normal cell death. However, estradiol at 100 nM significantly increased cell death relative to that detected in the control.

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US10/203,880 2000-02-15 2001-02-15 Cytoprotective steroids (II) Abandoned US20030092692A1 (en)

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AT (1) ATE358489T1 (fr)
AU (2) AU2001233856B2 (fr)
CA (1) CA2398706A1 (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080070879A1 (en) * 2006-09-14 2008-03-20 Sharon Sageman 7-keto dhea for psychiatric use
US20100160274A1 (en) * 2007-09-07 2010-06-24 Sharon Sageman 7-KETO DHEA for Psychiatric Use
WO2013097835A1 (fr) 2011-12-27 2013-07-04 Centro De Investigacion Y Desarrollo De Los Medicamentos (Cidem) Systèmes spiro-stéroïdiens ayant des effets neuroactifs et anti-inflammatoires

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GB2363983A (en) 2000-06-29 2002-01-16 Hunter Fleming Ltd Protection against neuronal damage using 7-hydroxyepiandrosterone
GB2363984A (en) * 2000-06-29 2002-01-16 Hunter Fleming Ltd Protection against neuronal damage using 3-hydroxy-7 -hydroxy steroids and 3-oxo-7 -hydroxy steroids
GB2378898A (en) * 2001-08-14 2003-02-26 Hunter Fleming Ltd Prophylactic and therapeutic use of hydroxysteroids
US7910755B2 (en) 2004-09-29 2011-03-22 Harbor Biosciences, Inc. Stem cell expansion and uses
EP2147673A1 (fr) 2006-11-30 2010-01-27 Hunter-Fleming Limited Modulation de chemins métaboliques de prostaglandine/cyclo-oxygénase
CZ301216B6 (cs) * 2008-07-10 2009-12-09 Ústav organické chemie a biochemie Akademie ved CR, v. v. i. Pregnanové anionické slouceniny, zpusob jejich výroby a jejich použití

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YU243975A (en) * 1974-10-14 1982-06-30 Schering Ag Process for obtaining 7-hydroxyestradiols
US5292730A (en) * 1990-08-29 1994-03-08 Humanetics Corporation Modulation of immune system with Δ5-androstenes
FR2696934B1 (fr) * 1992-10-20 1995-06-02 Conservatoire Nal Arts Metiers Dérivés de stéroïdes naturels 3B hydroxyles ayant des propriétés de déclenchement et de stimulation de l'immunité, composition les contenant et procédé pour les obtenir.
US5846963A (en) * 1995-06-07 1998-12-08 University Of Utah Research Foundation Methods for preventing progressive tissue necrosis, reperfusion injury, bacterial translocation and adult respiratory distress syndrome
US5554601A (en) * 1993-11-05 1996-09-10 University Of Florida Methods for neuroprotection
JP4313435B2 (ja) * 1995-07-24 2009-08-12 トラスティーズ オブ ボストン ユニバーシティー プレグネノロンサルフェート誘導体によるｎｍｄａレセプター活性の抑制
CA2250874A1 (fr) * 1996-04-09 1997-10-16 Btg International Limited Utilisation de steroides a substitution 7 alpha pour traiter les troubles neuropsychiatriques, immunitaires ou endocriniens
FR2760362B1 (fr) * 1997-03-10 2000-08-11 Vitasterol Utilisation cosmetique ou dermatologique de steroides 7-hydroxyles

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080070879A1 (en) * 2006-09-14 2008-03-20 Sharon Sageman 7-keto dhea for psychiatric use
US8124598B2 (en) 2006-09-14 2012-02-28 Sharon Sageman 7-keto DHEA for psychiatric use
US20100160274A1 (en) * 2007-09-07 2010-06-24 Sharon Sageman 7-KETO DHEA for Psychiatric Use
WO2013097835A1 (fr) 2011-12-27 2013-07-04 Centro De Investigacion Y Desarrollo De Los Medicamentos (Cidem) Systèmes spiro-stéroïdiens ayant des effets neuroactifs et anti-inflammatoires

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DE60127683D1 (de) 2007-05-16
WO2001060375A2 (fr) 2001-08-23
AU2001233856B2 (en) 2006-05-25
EP1307205A2 (fr) 2003-05-07
ATE358489T1 (de) 2007-04-15
JP2003522794A (ja) 2003-07-29
WO2001060375A8 (fr) 2001-11-15
EP1307205B1 (fr) 2007-04-04
GB0003524D0 (en) 2000-04-05
WO2001060375A3 (fr) 2002-04-04
CA2398706A1 (fr) 2001-08-23
AU3385601A (en) 2001-08-27

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